JP2017181368A - Immunoassay method, kit for immunoassay, and lateral-flow type chromatographic test strip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a virus or a bacterium with high sensitivity by immunological measurement. An influenza virus measurement kit for detecting or quantifying an influenza virus has a membrane 110 and a determination unit 130 in which a capture ligand 131 that specifically binds to the influenza virus 160 is immobilized on the membrane 110. A labeled antibody in which a lateral flow type chromatotest strip 200 containing the mixture and an antibody that specifically binds to influenza virus 160 are labeled with a resin-metal complex 100 having a structure in which a plurality of metal particles 20 are immobilized on the resin particles 10. Includes a detection reagent comprising 150 and. [Selection diagram] Fig. 1

Description

  The present invention relates to an immunoassay method capable of detecting and quantifying viruses and bacteria contained in a sample with high sensitivity, an immunoassay kit used therefor, and a lateral flow type chromatographic test strip.

  Since innumerable chemical substances exist in the living body, it is extremely important to analyze a specific trace component in the living body qualitatively and quantitatively. In the fields of medicine, pharmaceuticals, health food, biotechnology, environment, etc., drugs and foods that act only on specific parts (chemical substances) in the living body, analyzers and diagnostic agents that detect slight changes in the living body, It has been developed with the above technology.

  One of the analysis techniques is an immunoassay. This is also called an immunological measurement method, and is a method for qualitatively and quantitatively analyzing a trace component by utilizing a specific reaction between an antigen and an antibody, which is one of immune reactions. Antigen-antibody reaction is widely used in the above field because of its high sensitivity and selectivity of reaction. There are various measurement methods for immunoassays depending on the measurement principle. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), latex agglutination (LIA, PA), immunochromatography ( ICA), hemagglutination method (HA), hemagglutination inhibition method (HI) and the like.

  In the immunoassay, an antigen or antibody is detected qualitatively or quantitatively from a change (a change in the concentration of the antigen, antibody or complex) when the antigen reacts with the antibody to form a complex. When these are detected, the detection sensitivity is increased by binding a labeling substance to the antibody, antigen or complex. Therefore, it can be said that the labeling ability of the labeling substance is an important factor affecting the detection ability in the immunoassay. Also in the immunoassay exemplified above, as labeling substances, red blood cells (in the case of HA), latex particles (in the case of LIA), fluorescent dyes (in the case of FIA), radioactive elements (in the case of RIA), enzymes (in the case of EIA), Chemiluminescent materials (in the case of CLIA) are used.

  By the way, when colored fine particles are used as the labeling substance, detection can be confirmed by visual observation without using a special analyzer, so that simpler measurement is expected. As such colored fine particles, for example, Patent Document 1 proposes a colored latex composed of gold nanoparticles bonded to the surface of polymer latex particles. By bonding gold nanoparticles to the surface of the polymer latex particles, the gold nanoparticles themselves serve as a colorant to improve visibility and detection sensitivity, while the gold nanoparticles themselves have excellent binding properties to antigens or antibodies. Therefore, it is said that a sufficient amount of antigen or antibody can be bound even if gold nanoparticles are bound to such an extent that a sufficiently dark color is obtained.

  The colored latex is one in which gold nanoparticles are bonded to the surface of the latex by irradiating a dispersion of HAuCl, which is a precursor of styrene-acrylic acid copolymer latex and gold nanoparticles, with gamma rays. However, in the above colored latex, since gold nanoparticles are bound only to the surface of the latex, the amount of gold nanoparticles that exhibit surface plasmon absorption is limited and the gold nanoparticles are easily detached. As a result, the visibility and sensitivity as an immunological measurement reagent may not be sufficient. Moreover, since electromagnetic radiation such as gamma rays is irradiated, the latex may be damaged. Furthermore, in the specification of Patent Document 1, preferred ranges of the latex diameter and the gold nanoparticle diameter are disclosed. However, it is not clear whether these preferred ranges have been verified in the examples, and the preferred range is defined. There is no basis for.

  Patent Document 2 discloses polymer latex particles coated with metallic gold, and suggests application to reagents that can be used in microscopy and immunoassay methods. However, the polymer latex particles coated with metal gold do not disclose the material and particle size of the polymer latex particles. Furthermore, there is no verification of the effect as a reagent that can be used in immunoassay methods. Therefore, the effect as a reagent in metal gold and polymer latex particles is unknown.

  Against this background, the present inventors previously proposed a resin-metal complex having a specific structure as a labeling substance that enables highly sensitive immunological measurement (Patent Documents 3 and 4). , Japanese Patent Application No. 2015-139269).

  By the way, when detecting or quantifying an analyte contained in a biological sample such as blood, urine, saliva or a sample of food, a lateral flow type chromatographic test strip (hereinafter referred to as “test strip”) may be used. Analyte can be detected quickly and easily. Therefore, test strips are widely used in various clinical tests and certification tests. As an immunochromatograph using such a test strip, a so-called one-step type and two-step type are known.

  In the one-step type immunochromatograph, first, a sample containing an analyte is added to the sample addition section. The added sample moves (develops) on the membrane from the sample addition portion toward the determination portion by capillary action. Since the reaction part in the middle contains a labeled ligand that specifically binds to the analyte, the analyte binds to the labeled ligand, and a complex containing the analyte and the labeled ligand is formed. . Next, the formed complex moves to the determination unit. In the determination unit, a ligand (capture ligand) that specifically binds to the analyte in the complex is immobilized. Therefore, when the complex moves to the determination unit, the complex is captured by the determination unit via the binding reaction between the analyte in the complex and the capture ligand. Next, the determination unit detects a signal from the labeled substance in the captured complex, and qualitatively or quantitatively analyzes the analyte in the sample (for example, Patent Document 5).

  On the other hand, in the two-step type immunochromatograph, first, an analyte such as an antigen is specifically bound to the analyte, and a labeled ligand such as an antibody is contacted to form a complex. Next, this complex is added as a mobile phase to the test strip, developed on the membrane by the principle of chromatography, and the complex is captured by a capture ligand (for example, a second antibody against the antigen) at the reaction part of the membrane. Thereafter, a signal emitted from the label is detected, and the analyte in the sample is analyzed qualitatively or quantitatively (for example, Patent Document 6).

JP 2009-168495 A Japanese Patent Laid-Open No. 3-206959 International Publication WO2016 / 002742 International Publication WO2016 / 002743 JP 2011-27693 A JP 2005-522698 A

  In an immunochromatograph, sensitivity and visibility depend on the color density and the size of the particles. In order to increase the sensitivity, it is important that a small particle is seen by the test line even if it is caught by the test line. Therefore, a large particle is advantageous. Further, in order for a human to visually recognize the few particles, it is desirable to make the particles easy to see with dark colored particles. However, in immunochromatographs using conventional test strips, proteins containing enzymes, metal colloids, colored latex particles and the like have been generally used as labeling substances. Immunochromatographs using these labeling substances are not always satisfactory in terms of color developability and detection sensitivity, but have room for improvement.

For example, colloidal gold is a dark colored particle with high visibility, but many small particles with a particle size of 70 nm or less are used, so if the amount of antigen (such as virus) decreases, the visibility becomes extremely poor. , Sensitivity decreases. Further, if the size of the gold nanoparticles is increased to 100 nm or more in order to increase the sensitivity, the original metallic color of gold appears, the colloidal dyeing degree is remarkably lowered, and the visibility is lowered, so that it is difficult to increase the sensitivity.
On the other hand, colored latex particles are often 200 nm to 300 nm, and relatively large particles are often used. Therefore, even with a small amount of antigen, large particles can be caught by the test line, and an increase in sensitivity can be expected. However, there is a limit to the degree of dyeing of the latex itself, and it is not possible to obtain color development as deep as that of a gold colloid. Therefore, although the colored latex particles are large particles, there is a problem that visibility is deteriorated due to low dyeing degree and sensitivity cannot be increased. In addition, if the amount of dye is increased in order to increase the dyeing degree of the colored latex particles, the dispersibility of the particles is deteriorated, so that improvement of the dyeing degree cannot be expected.

  Therefore, an object of the present invention is to measure viruses and bacteria with high sensitivity by an immunological assay.

  As a result of intensive studies, the present inventors have found that by using a resin-metal complex having a specific structure as a labeling substance, excellent color development and detection sensitivity can be obtained, and the above problems can be solved. The headline and the present invention were completed.

That is, the immunoassay method of the present invention is a method for detecting or quantifying viruses or bacteria contained in a sample.
The immunoassay method of the present invention uses a lateral flow type chromatographic test strip comprising a membrane and a determination part in which a capture ligand that specifically binds to the virus is immobilized on the membrane, and the following steps (I) to (III) );
Step (I): The virus or bacteria contained in the sample and an antibody that specifically binds to the virus or bacteria are labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. A step of contacting the labeled antibody,
Step (II): A step of bringing the complex containing a virus or bacteria and a labeled antibody formed in Step (I) into contact with a capture ligand in the determination unit,
Step (III): a step of measuring a color intensity derived from light energy absorption by localized surface plasmon resonance and electronic transition of the resin-metal complex,
The process including this is performed.

  In the immunoassay method of the present invention, the metal particles may be gold particles or platinum particles.

  In the immunoassay method of the present invention, the metal particles in the resin-metal complex include first particles having portions exposed to the outside of the resin particles, and second particles entirely contained in the resin particles. And at least some of the first particles and the second particles may be three-dimensionally distributed in the surface layer portion of the resin particles.

  In the immunoassay method of the present invention, the resin particles may be polymer particles having a substituent in the structure capable of adsorbing metal ions.

  In the immunoassay method of the present invention, the average particle diameter of the metal particles may be in the range of 1 to 80 nm.

  In the immunoassay method of the present invention, the resin-metal complex may have an average particle size in the range of 50 to 1000 nm.

  In the immunoassay method of the present invention, the antibody may be an anti-influenza A monoclonal antibody.

The immunoassay kit of the present invention is an immunoassay kit for detecting or quantifying viruses or bacteria contained in a sample using a lateral flow type chromatographic test strip.
The immunoassay kit of the present invention includes a membrane, a lateral flow type chromatographic test strip including a determination part in which a capture ligand that specifically binds to the virus or bacteria is immobilized on the membrane, and the virus or bacteria. And a detection reagent comprising a labeled antibody labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles.

  The lateral flow type chromatographic test strip of the present invention is for detecting or quantifying viruses or bacteria contained in a sample. The lateral flow type chromatographic test strip of the present invention includes a membrane, a determination unit in which a capture ligand that specifically binds to the virus or bacteria is immobilized on the membrane in a direction in which the sample is developed, and the determination unit A reaction part containing a labeled antibody labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle, and an antibody that specifically binds to the virus or bacterium on the upstream side; ,including.

  According to the immunoassay method of the present invention, it is possible to achieve both excellent color development and high detection sensitivity by using a resin-metal complex labeled with an antibody, an antigen, or a protein, and blood, saliva, Antibodies, drugs, low molecular hormones (estrogens, etc.), high molecular hormones, cancer markers (stool hemoglobin, etc.), viruses (hepatitis B and C viruses, Influenza virus, etc.), bacteria (Staphylococcus aureus, Legionella, periodontal bacteria, etc.), high molecular hormones (insulin, etc.), DNA and RNA, cytokines, and other highly sensitive immunoassays. In addition, the immunoassay kit and lateral flow chromatographic test strip of the present invention are excellent in visibility, visual judgment and detection sensitivity, and are advantageous in immunological measurement of viruses and bacteria such as influenza virus and Legionella. Available.

It is explanatory drawing which shows the outline | summary of the immunoassay method using the lateral flow type | mold chromatographic test strip which concerns on one embodiment of this invention. It is a schematic diagram which shows the structure of the cross section of the resin-metal composite body used by one embodiment of this invention. 4 is a scanning electron microscope (SEM) photograph of the resin-metal composite obtained in Production Example 2.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.

[Lateral flow type chromatographic test strip]
First, a lateral flow type chromatographic test strip (test strip) according to an embodiment of the present invention will be described with reference to FIG. As will be described later, this test strip 200 can be preferably used in the immunoassay method of one embodiment of the present invention. Hereinafter, the case where influenza virus is measured will be described as an example.

  The test strip 200 includes a membrane 110. The membrane 110 is provided with a sample addition unit 120, a determination unit 130, and a liquid absorption unit 140 in order in the sample development direction.

<Membrane>
As the membrane 110 used in the test strip 200, a membrane used as a membrane material in a general test strip can be applied. The membrane 110 is formed of, for example, an inactive substance (a substance that does not react with the influenza virus 160, various ligands, etc.) made of a microporous material that exhibits capillary action and that simultaneously develops the sample. It is. Specific examples of the membrane 110 include a fibrous or non-woven fibrous matrix composed of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc., membrane, filter paper, glass fiber filter paper, cloth, Cotton etc. are mentioned. Among these, membranes composed of cellulose derivatives and nylon, filter paper, glass fiber filter paper, etc. are preferably used, more preferably nitrocellulose membrane, mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membrane, nylon membrane Filter paper is used.

  The test strip 200 is preferably provided with a support that supports the membrane 110 for easier operation. As the support, for example, plastic can be used.

<Sample addition part>
The test strip 200 may have a sample adding unit 120 for adding a sample containing the influenza virus 160. The sample addition unit 120 is a part for receiving a sample containing the influenza virus 160 in the test strip 200. The sample addition unit 120 may be formed on the membrane 110 upstream of the determination unit 130 in the direction in which the sample is developed, or, for example, cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon A sample addition pad made of a material such as cotton cloth may be provided on the membrane 110 to constitute the sample addition unit 120.

<Determining unit>
A capture ligand 131 that specifically binds to the influenza virus 160 is fixed to the determination unit 130. The capture ligand 131 can be used without particular limitation as long as it forms a specific bond with the influenza virus 160. For example, an antibody against the influenza virus 160 can be preferably used. The capture ligand 131 is immobilized so as not to move from the determination unit 130 even when a sample is provided to the test strip 200. The capture ligand 131 may be fixed directly or indirectly to the membrane 110 by physical or chemical bonding or adsorption.

  The determination unit 130 is not particularly limited as long as the complex 170 including the labeled antibody 150 and the influenza virus 160 is in contact with the capture ligand 131 that specifically binds to the influenza virus 160. For example, the capture ligand 131 may be directly fixed to the membrane 110, or the capture ligand 131 may be fixed to a pad made of cellulose filter paper, glass fiber, nonwoven fabric, or the like fixed to the membrane 110. .

<Liquid absorption part>
The liquid-absorbing part 140 is formed by a pad of a water-absorbing material such as cellulose filter paper, non-woven fabric, cloth or cellulose acetate, for example. The moving speed of the sample after the development front (front line) of the added sample reaches the liquid absorption part 140 varies depending on the material, size, and the like of the liquid absorption part 140. Therefore, the optimum speed for detecting and quantifying the influenza virus 160 can be set by selecting the material, size, and the like of the liquid absorption part 140. In addition, the liquid absorption part 140 is an arbitrary configuration and may be omitted.

  The test strip 200 may further include arbitrary parts such as a reaction part and a control part as necessary.

<Reaction part>
Although not shown, the test strip 200 may be formed with a reaction part including the labeled antibody 150 on the membrane 110. The reaction unit can be provided upstream of the determination unit 130 in the direction in which the sample flows. In addition, you may utilize the sample addition part 120 in FIG. 1 as a reaction part. When the test strip 200 has a reaction part, when the sample containing the influenza virus 160 is subjected to the reaction part or the sample addition part 120, the influenza virus 160 and the labeled antibody 150 contained in the sample may be brought into contact with each other in the reaction part. it can. In this case, since the complex 170 containing the influenza virus 160 and the labeled antibody 150 can be formed by simply supplying the sample to the reaction part or the sample addition part 120, so-called one-step type immunochromatography is possible. Become.

  The reaction part is not particularly limited as long as it includes the labeled antibody 150 that specifically binds to the influenza virus 160, but may be one in which the labeled antibody 150 is directly applied to the membrane 110. Alternatively, the reaction part may be formed by, for example, immobilizing the membrane 110 with a pad (conjugate pad) made of cellulose filter paper, glass fiber, nonwoven fabric, or the like impregnated with the labeled antibody 150.

<Control part>
Although illustration is omitted, the test strip 200 may be formed with a control unit in which a capture ligand that specifically binds to the labeled antibody 150 is fixed to the membrane 110 in the direction in which the sample is developed. By measuring the color intensity at the control unit together with the determination unit 130, the sample provided for the test strip 200 is developed and reaches the reaction unit and the determination unit 130 to confirm that the test has been performed normally. be able to. The control unit is created in the same manner as the determination unit 130 described above except that another type of capture ligand that specifically binds to the labeled antibody 150 is used instead of the capture ligand 131. The configuration can be taken. In addition to the above-described method, the control unit may be a substance showing a key-keyhole relationship, such as avidin and biotin, an enzyme and a substrate, an enzyme and a coenzyme. Preferably, avidin and biotin may be used in place of the capture ligand 131 and the labeled antibody 150.

[Measurement method of influenza virus]
Next, a method for measuring influenza virus 160 according to an embodiment of the present invention performed using test strip 200 will be described.

The method for measuring influenza virus 160 according to the present embodiment is a method for measuring influenza virus 160 for detecting or quantifying influenza virus 160 contained in a sample. Here, the influenza virus 160 includes not only a human but also a virus that infects non-human animals such as an avian influenza virus. In addition, the influenza virus 160 may be any antigen as long as it has antigenicity, and may be a part of the virus, for example, NP antigen, M1 antigen, HA antigen, NA antigen, and the like. The method for measuring influenza virus 160 according to the present embodiment uses a test strip 200 including a membrane 110 and a determination unit 130 in which a capture ligand 131 that specifically binds to the influenza virus 160 is immobilized on the membrane 110. Steps (I) to (III);
Step (I): The influenza virus 160 contained in the sample and an antibody that specifically binds to the influenza virus 160 are labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. Contacting the labeled antibody 150,
Step (II): a step of bringing the complex containing the influenza virus 160 and the labeled antibody 150 formed in Step (I) into contact with the capture ligand 131 in the determination unit 130;
Step (III): a step of measuring the color intensity derived from light energy absorption by localized surface plasmon resonance and electronic transition of the resin-metal complex,
Can be included.

Process (I):
Step (I) is a step of bringing the influenza virus 160 contained in the sample into contact with the labeled antibody 150. As long as the complex 170 containing the influenza virus 160 and the labeled antibody 150 is formed, the mode of contact is not particularly limited. For example, the sample may be provided to the sample addition unit 120 or the reaction unit (not shown) of the test strip 200, and the influenza virus 160 may be contacted with the labeled antibody 150 in the reaction unit, or before the sample is applied to the test strip 200. The influenza virus 160 in the sample may be brought into contact with the labeled antibody 150.

  The composite 170 formed in the step (I) expands and moves on the test strip 200 and reaches the determination unit 130.

Process (II):
In step (II), the determination unit 130 of the test strip 200 causes the complex 170 containing the influenza virus 160 and the labeled antibody 150 formed in step (I) to contact the capture ligand 131. When the complex 170 is brought into contact with the capture ligand 131, the capture ligand 131 specifically binds to the influenza virus 160 of the complex 170. As a result, the complex 170 is captured by the determination unit 130.

  Since the capture ligand 131 does not specifically bind to the labeled antibody 150, when the influenza virus 160 and the unbound labeled antibody 150 reach the determination unit 130, the labeled antibody 150 unbound to the influenza virus 160. Passes through the determination unit 130. Here, when a control unit (not shown) to which another capture ligand that specifically binds to the labeled antibody 150 is fixed is formed on the test strip 200, the labeled antibody 150 that has passed through the determination unit 130 is developed. To bind to the other capture ligand at the control unit. As a result, the labeled antibody 150 that does not form the complex 170 with the influenza virus 160 is captured by the control unit.

  After step (II), if necessary, before step (III), for example, the test strip 200 is washed with a buffer solution commonly used in biochemical tests such as water, physiological saline, and phosphate buffer solution. A cleaning step may be performed. The washing step removes the labeled antibody 150 (the labeled antibody 150 that is not bound to the influenza virus 160 and does not form the complex 170) that has not been captured by the judging unit 130 or the judging unit 130 and the control unit. be able to.

  By performing the washing process, in Step (III), the coloration due to light energy absorption by the determination surface 130 or the localized surface plasmon resonance and electronic transition of the resin-metal complex in the determination portion 130 and the control portion is measured. In this case, the color intensity of background can be reduced, the signal / background ratio can be increased, and the detection sensitivity and quantitativeness can be further improved.

Process (III):
Step (III) is a step of measuring the color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transition of the resin-metal complex. After performing the above step (II) or the cleaning step as necessary, the test strip 200 measures the color intensity derived from the light energy absorption due to localized surface plasmon resonance and electronic transition of the resin-metal composite.

  When the control part is formed on the test strip 200, the labeled antibody 150 is captured by another capture ligand in the control part in the step (II) to form a complex. Therefore, in the step (III), in the test strip 200, not only the determination unit 130 but also the control unit can generate color by light energy absorption due to localized surface plasmon resonance and electronic transition. In this way, by measuring the color intensity in the control unit as well as the determination unit 130, it is possible to confirm whether the sample provided for the test strip 200 has developed normally and reached the reaction unit and the determination unit 130.

<Sample>
The sample in the method for measuring influenza virus 160 according to the present embodiment is not particularly limited as long as it contains influenza virus 160. For example, a biological sample containing the target influenza virus 160 (ie whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swab, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin exudate , Extracts from tissues, cells and stool, etc.). If necessary, in order to facilitate the specific binding reaction between the labeled antibody 150 and the capture ligand 131 and the influenza virus 160, the influenza virus 160 contained in the sample is pretreated prior to the step (I). May be. Here, examples of the pretreatment include chemical treatment using various chemicals such as acid, base, and surfactant, and physical treatment using heating, stirring, ultrasonic waves, and the like. In particular, in the case of a substance that is not normally exposed on the surface, such as an influenza virus NP antigen, treatment with a surfactant or the like is preferable. As a surfactant used for this purpose, a nonionic surfactant can be used in consideration of specific binding reaction, for example, binding reactivity between a ligand such as an antigen-antibody reaction and influenza virus 160. .

  In addition, the sample may be appropriately diluted with a solvent (water, physiological saline, buffer, or the like) used in a normal immunological analysis method or a water-miscible organic solvent.

<Labeled antibody>
In the step (I), the labeled antibody 150 is used to contact the influenza virus 160 contained in the sample to form a complex 170 containing the analyte influenza virus 160 and the labeled antibody 150. The labeled antibody 150 is obtained by labeling an antibody that specifically binds to the influenza virus 160 with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. Here, “labeling” means that the resin-metal complex is directly or indirectly attached to the antibody to the extent that the resin-metal complex is not detached from the labeled antibody 150 in steps (I) to (III). Furthermore, it means that it is fixed by chemical or physical bonding or adsorption. For example, the labeled antibody 150 may be one in which a resin-metal complex is directly bonded to the antibody, or one in which the antibody and the resin-metal complex are bonded via an arbitrary linker molecule. Alternatively, each may be fixed to insoluble particles.

  In the present embodiment, the resin-metal composite used as a label has a structure in which a plurality of metal particles are immobilized on resin particles. The details of the resin-metal composite will be described later.

In the present embodiment, the “antibody” is not particularly limited. For example, in addition to a polyclonal antibody, a monoclonal antibody, an antibody obtained by genetic recombination, an antibody fragment [for example, an H chain , L chain, Fab, Fab ′, F (ab ′) ₂ , SCFV, etc.] can be used. Specific examples of preferred antibodies include anti-influenza virus antibodies such as anti-influenza A monoclonal antibodies, anti-influenza B monoclonal antibodies, and anti-influenza C monoclonal antibodies, and anti-legionella polyclonal antibodies.

<Preferred production method of labeled antibody>
Next, a preferred method for producing the labeled antibody 150 will be described. The production of the labeled antibody 150 is at least the following step A;
Step A) including the step of obtaining the labeled antibody 150 by mixing and binding the resin-metal complex with the antibody under a first pH condition, and preferably, further includes Step B;
Step B) A step of treating the labeled antibody 150 under a second pH condition can be included.

[Step A]
In step A, the labeled antibody 150 is obtained by mixing the resin-metal complex with the antibody under the first pH condition. In step A, it is preferable to contact the antibody in a state where the solid resin-metal complex is dispersed in the liquid phase. The first pH condition varies depending on the metal species of the metal particles in the resin-metal composite.

  When the metal particles of the resin-metal composite are gold particles (including gold alloy particles; the same applies hereinafter), the first pH condition can be applied under any of acidic, neutral, and alkaline conditions. However, from the viewpoint of uniformly contacting the resin-metal complex and the antibody while maintaining the dispersion of the resin-metal complex and the activity of the antibody, conditions within the range of pH 2 to 7 are preferable, and acidic conditions such as pH 2. A range of 5 to 5.5 is more preferable. When the metal particles are gold particles, the resin-metal complex and the antibody may be deactivated due to strong acidity when the pH is less than 2, and the resin-metal complex is inactivated when the pH exceeds 7. When the body and antibody are mixed, they aggregate and become difficult to disperse. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.

  Further, when the metal particles of the resin-metal complex are particles other than gold, such as platinum, palladium, or an alloy thereof, the first pH condition is that the dispersion of the resin-metal complex and the activity of the antibody are controlled. From the viewpoint of bringing the resin-metal complex and antibody into uniform contact with each other while being maintained, conditions within a pH range of 2 to 10 are preferable, and for example, a pH range of 5 to 9 is more preferable. When the metal particles are particles other than gold, when the resin-metal complex and the antibody are bound under a pH of less than 2, the antibody may be denatured and deactivated due to strong acidity. When the metal complex and the antibody are mixed, they aggregate and become difficult to disperse. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.

  Step A is preferably performed in a binding buffer adjusted to the first pH condition. For example, a predetermined amount of the resin-metal complex is mixed with the binding buffer adjusted to the above pH and mixed thoroughly. As the binding buffer, for example, a boric acid solution adjusted to a predetermined concentration can be used. The pH of the binding buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide.

  Next, a labeled antibody-containing liquid can be obtained by adding a predetermined amount of the antibody to the obtained mixed liquid, sufficiently stirring and mixing. The labeled antibody-containing liquid thus obtained can be fractionated only with the labeled antibody 150 as a solid part by solid-liquid separation means such as centrifugation.

[Step B]
In the process B, the labeled antibody 150 obtained in the process A is treated under the second pH condition to perform blocking that suppresses nonspecific adsorption to the labeled antibody 150. In this case, the labeled antibody 150 separated by the solid-liquid separation means is dispersed in the liquid phase under the second pH condition. This blocking condition varies depending on the metal species of the metal particles in the resin-metal composite.

  When the metal particles of the resin-metal complex are gold particles, the second pH condition is preferably within the range of pH 2 to 9, for example, from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150. From the viewpoint of suppressing non-specific adsorption of the labeled antibody 150, acidic conditions, for example, in the range of pH 2 to 6, are more preferable. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if it exceeds pH 9, the labeled antibody 150 aggregates, making dispersion difficult.

  Further, when the metal particles of the resin-metal complex are particles other than gold, the second pH condition is, for example, from pH 2 to 10 from the viewpoint of maintaining the activity of the antibody and suppressing aggregation of the labeled antibody 150. Within the range, from the viewpoint of suppressing nonspecific adsorption of the labeled antibody 150, the range of pH 5 to 9 is more preferable. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if it exceeds pH 10, the labeled antibody 150 aggregates, making dispersion difficult.

  The step B is preferably performed using a blocking buffer adjusted to the second pH condition. For example, the blocking buffer adjusted to the above pH is added to a predetermined amount of labeled antibody 150, and the labeled antibody 150 is uniformly dispersed in the blocking buffer. As the blocking buffer, for example, a protein solution that does not bind to the detection target is preferably used. Examples of proteins that can be used in the blocking buffer include bovine serum albumin, ovalbumin, casein, and gelatin. In addition to proteins, artificial polymer polymers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and amino acid polymers may be used. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. A known method can be used for dispersing the labeled antibody 150. For example, it is preferable to use a dispersing means such as ultrasonic treatment or stirring by a rotator. In this way, a dispersion in which the labeled antibody 150 is uniformly dispersed is obtained.

  A dispersion of labeled antibody 150 is obtained as described above. From this dispersion, only the labeled antibody 150 can be fractionated as a solid part by solid-liquid separation means such as centrifugation. Moreover, a cleaning process, a preservation | save process, etc. can be implemented as needed. Hereinafter, the cleaning process and the storage process will be described.

(Cleaning process)
In the washing treatment, a washing buffer solution is added to the labeled antibody 150 sorted by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the washing buffer solution. For dispersion, for example, a dispersion means such as ultrasonic treatment is preferably used. The washing buffer is not particularly limited, but for example, a Tris buffer solution, a glycinamide buffer solution, an arginine buffer solution, or the like having a predetermined concentration adjusted within a pH range of 8 to 9 may be used. it can. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. The washing treatment of the labeled antibody 150 can be repeated a plurality of times as necessary.

(Save process)
In the storage process, a storage buffer is added to the labeled antibody 150 collected by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the storage buffer. For dispersion, for example, a dispersion means such as ultrasonic treatment is preferably used. As the storage buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregation agent and / or stabilizer to the washing buffer can be used. Examples of the aggregation inhibitor include saccharides represented by sucrose, maltose, lactose, trehalose, artificial polymers such as glycerin, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and polyethylene glycol (PEG), octylphenol ethoxylate. And nonionic surfactants such as polysorbates, ionic surfactants, and the like can be used. The stabilizer is not particularly limited. For example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin, and saccharides represented by sucrose, maltose, lactose, and trehalose can be used. Thus, the preservation | save process of the labeled antibody 150 can be performed.

  In each of the above steps, a surfactant or a preservative such as sodium azide or paraoxybenzoate can be used as necessary.

<Resin-metal composite>
Next, the resin-metal complex used as a label in the method for measuring influenza virus of the present embodiment will be described in detail. FIG. 2 is a schematic cross-sectional view of a resin-metal composite used as a label in the present embodiment. The resin-metal composite 100 includes resin particles 10 and metal particles 20.

  In the resin-metal composite 100, the metal particles 20 are dispersed or immobilized on the resin particles 10. In the resin-metal composite 100, a part of the metal particles 20 is three-dimensionally distributed in the surface layer portion 60 of the resin particle 10, and a part of the three-dimensionally distributed metal particles 20 is partially. The resin particles 10 are exposed outside, and the remaining part is encapsulated in the resin particles 10.

  Here, the metal particles 20 include metal particles completely encapsulated in the resin particles 10 (hereinafter also referred to as “encapsulated metal particles 30”), portions embedded in the resin particles 10, and outside the resin particles 10. Metal particles having an exposed portion (hereinafter also referred to as “partially exposed metal particles 40”) and metal particles adsorbed on the surfaces of the resin particles 10 (hereinafter also referred to as “surface-adsorbed metal particles 50”). Exists. The partially exposed metal particles 40 and the surface-adsorbed metal particles 50 correspond to “first particles” in the present invention, and the encapsulated metal particles 30 correspond to “second particles” in the present invention.

  For example, when the resin-metal complex 100 is used for immunological measurement, the antibody is immobilized on the partially exposed metal particles 40 or the surface-adsorbed metal particles 50 and used. At this time, the antibody is immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, while not immobilized on the encapsulated metal particles 30. However, since all of the metal particles 20 including the encapsulated metal particles 30 exhibit absorption by localized surface plasmon resonance and light energy absorption by electronic transition, not only the partially exposed metal particles 40 and the surface adsorbed metal particles 50 are used. The encapsulated metal particles 30 also contribute to the improvement of visibility as a label for immunological measurement. Further, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a large contact area with the resin particles 10 as compared with the surface-adsorbed metal particles 50, and also have a physical adsorption force such as an anchor effect due to the embedded state. Strong and difficult to desorb from the resin particles 10. Therefore, the durability and stability as an immunological measurement label using the resin-metal complex 100 can be improved.

  The inner metal particles 30 are all covered with the resin constituting the resin particles 10 on the entire surface. Further, the partially exposed metal particles 40 are those in which 5% or more and less than 100% of the surface area is covered with the resin constituting the resin particles 10. From the viewpoint of durability for each of the above uses, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. Further, the surface-adsorbed metal particles 50 are covered with the resin constituting the resin particles 10 with more than 0% and less than 5% of the surface area thereof.

  In addition, the loading amount of the metal particles 20 (the total of the encapsulated metal particles 30, the partially exposed metal particles 40 and the surface-adsorbed metal particles 50) on the resin-metal composite 100 is based on the weight of the resin-metal composite 100. It is preferable that it is 5 wt%-70 wt%. If it is this range, the resin-metal composite body 100 is excellent in the visibility as a labeling substance, visual judgment property, and detection sensitivity. When the loading amount of the metal particles 20 is less than 5 wt%, the amount of antibody immobilized becomes small, and the detection sensitivity tends to decrease. More preferably, the supported amount of the metal particles 20 is 15 wt% to 70 wt%.

  Further, 10 wt% to 90 wt% of the metal particles 20 are preferably the partially exposed metal particles 40 and the surface adsorbed metal particles 50. If it is this range, since the fixed quantity of the antibody on the metal particle 20 can fully be ensured, the sensitivity as a labeling substance is high. More preferably, 20 wt% to 80 wt% of the metal particles 20 are the partially exposed metal particles 40 and the surface adsorbed metal particles 50, and the surface adsorbed metal particles 50 are more preferably 20 wt% or less from the viewpoint of durability. .

  Further, 60 wt% to 100 wt% of the metal particles 20 are present in the surface layer portion 60, and 5 wt% to 90 wt% of the metal particles 20 existing in the surface layer portion 60 are partially exposed metal particles 40 or surface adsorbed metal particles 50. It is preferable that the amount of the antibody immobilized on the metal particles 20 can be sufficiently secured, so that the sensitivity as a labeling substance is increased. In other words, it is preferable that 10 wt% to 95 wt% of the metal particles 20 existing in the surface layer portion 60 are the encapsulated metal particles 30.

  Here, the “surface layer portion” means a range of 50% of the particle radius in the depth direction from the surface of the resin particle 10. The “three-dimensional distribution” means that the metal particles 20 are dispersed not only in the surface direction of the resin particles 10 but also in the depth direction.

The resin particles 10 are preferably polymer particles having a substituent in the structure capable of adsorbing metal ions. In particular, nitrogen-containing polymer particles are preferable. Nitrogen atoms in the nitrogen-containing polymer are preferable because they easily chemisorb anionic metal ions that are precursors of the metal particles 20 such as gold, platinum, and palladium, which are excellent in visibility and can be easily immobilized. In the present embodiment, metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, so that part of the generated metal particles 20 becomes encapsulated metal particles 30 or partially exposed metal particles 40. . Moreover, since carboxylic acid etc. can adsorb | suck a cationic metal ion like an acrylic acid polymer, it is easy to adsorb | suck the cationic metal ion which is a precursor of the metal particles 20, such as silver, nickel, copper, Metal particles 20 such as silver, nickel, and copper can be formed, and an alloy with the metal such as gold, platinum, and palladium can be formed.
On the other hand, in the case of resin particles other than the nitrogen-containing polymer having a substituent capable of adsorbing metal ions, such as polystyrene, it is difficult to adsorb the metal ions inside the resin. As a result, most of the generated metal particles 20 become surface-adsorbed metal particles 50. As described above, since the surface-adsorbed metal particles 50 have a small contact area with the resin particles 10, the adhesive force between the resin and the metal is small, and the influence of the metal particles 20 being detached from the resin particles 10 tends to be large.
The nitrogen-containing polymer is a resin having a nitrogen atom in the main chain or side chain, and examples thereof include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, and polyaniline. Preferable are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. Moreover, when it has a nitrogen atom in a side chain, for example, an acrylic resin, a phenol resin, an epoxy resin, etc. can be used widely.

  As a material of the metal particles 20, for example, silver, nickel, copper, gold, platinum, palladium or the like can be applied. Gold, platinum and palladium are preferable because they are excellent in visibility and can be easily immobilized. These are preferred because they express absorption derived from light energy by localized surface plasmon resonance and electronic transition. More preferably, it is gold with good storage stability. These metals can be used as a simple substance or a composite such as an alloy. Further, from the viewpoint that excellent color developability can be obtained when the resin-metal complex 100 is bound to an antibody under the condition of pH 2 to 7, gold, gold alloy, platinum, Alternatively, platinum alloys are the most preferred metal species. Here, the gold alloy means, for example, an alloy made of gold and a metal species other than gold and containing 10% by weight or more of gold. Here, the other metal species forming the alloy with gold is not particularly limited, but for example, silver, nickel, copper, platinum, palladium and the like are preferable, and platinum, palladium and the like having excellent storage stability and visibility are more preferable. preferable. The platinum alloy means an alloy made of platinum and a metal species other than platinum and containing 1 wt% or more of platinum. Here, the other metal species forming the alloy with platinum is not particularly limited, but, for example, silver, nickel, copper, gold, palladium, and the like are preferable, and gold, palladium, and the like excellent in storage stability and visibility are more preferable. preferable.

  Moreover, it is preferable that the average particle diameter of the metal particle 20 measured by scanning electron microscope (SEM) observation is, for example, 1 to 80 nm from the viewpoint of obtaining high detection sensitivity in immunological measurement. When the average particle diameter of the metal particles 20 is less than 1 nm or exceeds 80 nm, the sensitivity tends to decrease because absorption due to localized surface plasmon resonance and light energy absorption due to electronic transition are difficult to be expressed. For example, in the case of gold particles, the average particle diameter of the metal particles 20 is preferably 20 nm or more and less than 70 nm, and more preferably 22 nm or more and less than 50 nm, from the viewpoint of obtaining high detection sensitivity in immunological measurement. Further, for example, in the case of platinum particles, from the viewpoint of obtaining high detection sensitivity in immunological measurement, it is preferably 1 nm to 50 nm, more preferably 1 nm to 30 nm, still more preferably 1 nm to 20 nm, Most preferably, it is 1 nm or more and 15 nm or less. In particular, when the average particle diameter of the platinum particles is 15 nm or less, extremely excellent detection sensitivity can be obtained when the resin-metal complex 100 is used as a labeling substance for immunochromatography.

  Moreover, it is preferable that the average particle diameter of the resin-metal composite body 100 is 50-1000 nm, for example. If the average particle size is less than 50 nm, for example, the amount of metal supported tends to be small, and thus the color tends to be weaker than metal particles of the same size. There is a tendency that the pores of a chromatographic medium such as a membrane filter are easily clogged, and the dispersibility tends to be lowered. The average particle diameter of the resin-metal composite 100 is preferably 100 nm or more and less than 700 nm, more preferably 250 nm or more and 600 nm or less, and most preferably 300 nm or more and 600 nm or less. In particular, when the average particle size is 300 nm or more, stable and excellent detection sensitivity can be obtained when used as a labeling substance for immunochromatography. Here, the particle size of the resin-metal composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed metal particle 40 or the surface-adsorbed metal particle 50 to the particle size of the resin particle 10, and laser diffraction. / Measured by scattering method, dynamic light scattering method, or centrifugal sedimentation method.

  The manufacturing method of the resin-metal composite 100 is not particularly limited. For example, a solution containing metal ions is added to a dispersion of resin particles 10 produced by an emulsion polymerization method, and the metal ions are adsorbed on the resin particles 10 (hereinafter referred to as “metal ion adsorption resin particles”). Furthermore, the metal ion adsorption resin particles are added to the reducing agent solution to reduce the metal ions to generate the metal particles 20, thereby obtaining the resin-metal composite 100.

Further, for example, when gold particles are used as the metal particles 20, examples of the solution containing metal ions include chloroauric acid (HAuCl ₄ ) aqueous solution. Moreover, you may use a metal complex instead of a metal ion.
Further, as a solvent for a solution containing metal ions, instead of water, water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, hydrochloric acid, sulfuric acid, nitric acid An acid such as the above may be used.
Further, in the solution, if necessary, for example, water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether; alkylene glycol, polyalkylene glycol, these Additives such as various water-miscible organic solvents such as polyols such as monoalkyl ether or dialkyl ether, glycerol, etc .; ketones such as acetone and methyl ethyl ketone may be added. Such an additive is effective for accelerating the reduction reaction rate of metal ions and controlling the size of the metal particles 20 to be produced.

Moreover, a well-known thing can be used for a reducing agent. For example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, Rochelle salt, etc. Can be mentioned. Of these, sodium borohydride, dimethylamine borane, and citric acid are preferable. If necessary, a surfactant can be added to the reducing agent solution, and the pH of the solution can be adjusted. The pH can be adjusted with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
Furthermore, the particle size of the metal particles 20 to be formed can be controlled by adjusting the reduction rate of the metal ions according to the temperature of the reducing agent solution.

  Further, when the metal ions in the metal ion adsorption resin particles are reduced to produce the metal particles 20, the metal ion adsorption resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion adsorption resin. Although it may be added to the particles, the former is preferable from the viewpoint of easy generation of the encapsulated metal particles 30 and the partially exposed metal particles 40.

  In order to maintain the dispersibility of the resin-metal composite 100 in water, for example, citric acid, poly-L-lysine, polyvinyl pyrrolidone, polyvinyl pyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemy Japan) A dispersant may be added. Furthermore, the dispersibility can be maintained by adjusting the pH with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.

  The resin-metal complex 100 having the above configuration can be preferably applied to immunoassay of viruses and bacteria by adsorbing antibodies on the surface of the metal particles 20 in particular. In particular, a labeling substance for immunological measurement of an influenza virus or a reagent for immunological measurement that is excellent in visual judgment property even in a low concentration region (high sensitivity region) and can achieve both excellent visibility and high detection sensitivity. It can be preferably applied as a material. The form of the immunological measurement labeling substance or immunological measurement reagent is not particularly limited. For example, a dispersion in which the resin-metal complex 100 is dispersed in water or a pH-adjusted buffer. Can be used as

[Immunodetection / quantification kit]
The immunoassay kit according to one embodiment of the present invention uses, for example, a lateral flow type chromatographic test strip 200 to detect the influenza virus 160 contained in a sample based on the influenza virus measurement method of the present embodiment. Alternatively, it is a kit for quantification.

The immunoassay kit of the present embodiment is
A membrane 110;
A lateral flow type chromatographic test strip 200 including a determination unit 130 in which a capture ligand 131 that specifically binds to the influenza virus 160 is immobilized on the membrane 110;
A detection reagent comprising a labeled antibody 150 in which an antibody that specifically binds to influenza virus 160 is labeled with a resin-metal complex 100 having a structure in which a plurality of metal particles 20 are immobilized on resin particles 10;
Is included. The immunoassay kit of the present embodiment may further include other components as necessary.

  In using the immunoassay kit according to the present invention, the influenza virus 160 in the sample and the labeled antibody 150 in the detection reagent are brought into contact with each other, and then the step (I) is performed. A sample may be provided to the adding unit 120, and the step (II) and the step (III) may be sequentially performed. Alternatively, after a detection reagent is applied to the upstream side of the determination unit 130 of the test strip 200 and appropriately dried to form a reaction part, the formed reaction part or a position upstream of the reaction part (for example, The sample may be added to the sample addition unit 120) and the steps (I) to (III) may be sequentially performed.

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are as follows.

<Measurement of absorbance of resin-metal composite>
Absorbance of the resin-metal composite was measured by placing a resin-metal composite dispersion (dispersion medium: water) prepared at 0.01 wt% into an optical white glass cell (optical path length 10 mm), and an instantaneous multi-photometry system (Otsuka The absorbance at 570 nm for gold and 700 nm for platinum was measured using MCPD-3700 manufactured by Denki Co., Ltd. In the case of gold, the absorbance at 570 nm was 0.9 (good), 0.5 to less than 0.9 was Δ (good), and less than 0.5 was x (impossible). In the case of platinum, the absorbance at 700 nm was 0.6 (good), 0.4 to less than 0.6 was Δ (good), and less than 0.4 was x (impossible).

<Measurement of solid content concentration and metal loading>
1 g of the dispersion before concentration adjustment was placed in a magnetic crucible, and heat treatment was performed at 70 ° C. for 3 hours. The weight before and after the heat treatment was measured, and the solid content concentration was calculated by the following formula.

  Solid content concentration (wt%) = [weight after drying (g) / weight before drying (g)] × 100

The sample after the heat treatment was further heat-treated at 500 ° C. for 5 hours, the weight before and after the heat treatment was measured, and the metal loading was calculated from the following formula.
Metal loading (wt%) =
[Weight after heat treatment at 500 ° C. (g) / Weight before heat treatment at 500 ° C. (g)] × 100

<Measurement of average particle diameter of resin-metal composite>
Measurement was performed using a disk centrifugal particle size distribution meter (manufactured by CPS Instruments). The measurement was performed in a state where the resin-metal composite was dispersed in water.

<Measurement of average particle diameter of metal particles>
The average particle size of the metal particles was measured by dropping a resin-metal composite dispersion liquid onto a metallic mesh with a carbon support film, using a field emission scanning electron microscope (STEM; manufactured by Hitachi High-Technologies Corporation, SU -9000), the area average diameter of the metal particles was measured.

[Production Example 1]
<Synthesis of resin particles>
Aliquat 336 (manufactured by Aldrich) (2.00 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 48.00 g) and Divinylbenzene (DVB, 2.00 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g) dissolved in 18.00 g of pure water was added dropwise and stirred at 60 ° C. for 3.5 hours. Resin particles A-1 having an average particle size of 0.38 μm were obtained. After sedimentation by centrifugation and removal of the supernatant, the operation of redispersing in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-1.

<Synthesis of resin-metal composite>
After adding 1233 ml of pure water to the resin particle dispersion B-1 (50 ml), a 30 mM aqueous solution of chloroauric acid (100 ml) was added and left at room temperature for 24 hours. Thereafter, the resin particles were precipitated by centrifugation and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-1.
Next, the above C-1 (42.4 ml) was added to 1580 ml of pure water, 528 mM dimethylamine borane aqueous solution (10 ml) was added dropwise with stirring at 3 ° C., and the mixture was stirred at room temperature for 2 hours. A resin-gold composite D-1 having a particle size of 0.399 μm was obtained. The D-1 is precipitated by centrifugation, the supernatant is removed, and the operation of redispersing in pure water is repeated three times, followed by purification by dialysis, whereby a 1 wt% resin-gold complex dispersion E -1 was obtained. The absorbance of the resin-gold complex F-1 in E-1 was 0.98 as a result of measurement according to the above method. Moreover, the average particle diameter of the gold particles in F-1 was 25.1 nm, and the amount of gold supported was 53.2 wt%.

[Production Example 2]
<Synthesis of resin particles>
Aliquat 336 (manufactured by Aldrich) (1.00 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 48.00 g) and Divinylbenzene (DVB, 2.00 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g) dissolved in 18.00 g of pure water was added dropwise over 2 minutes and stirred at 60 ° C. for 3.5 hours. Thus, resin particles A-2 having an average particle size of 0.42 μm were obtained. After sedimentation by centrifugation and removal of the supernatant, the operation of redispersing in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-2.

<Synthesis of resin-metal composite>
After adding 1233 ml of pure water to the resin particle dispersion B-2 (50 ml), a 30 mM chloroauric acid aqueous solution (100 ml) was added and left at room temperature for 24 hours. Thereafter, the resin particles were precipitated by centrifugation and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare 2.5 wt% gold ion adsorption resin particle dispersion C-2.
Next, the above-mentioned C-2 (42.4 ml) was added to 1580 ml of pure water, 528 mM dimethylamine borane aqueous solution (10 ml) was added dropwise with stirring at 3 ° C., and then stirred at room temperature for 2 hours. A resin-gold composite D-2 having a particle size of 0.438 μm was obtained. The D-2 is precipitated by centrifugation, the supernatant is removed, and the operation of redispersing in pure water is repeated three times, followed by purification by dialysis, whereby a 1 wt% resin-gold complex dispersion E -2 was obtained. The absorbance of the resin-gold complex F-2 in E-2 was 0.98 as a result of measurement according to the above method. Moreover, the average particle diameter of the gold particles in F-2 was 25.0 nm, and the amount of gold supported was 54.7 wt%. A scanning electron microscope (SEM) photograph of F-2 is shown in FIG.

[Production Example 3]
Aliquat 336 (manufactured by Aldrich) (3.00 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 49.50 g) and Divinylbenzene (DVB, 0.50 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.250 g) dissolved in 18.00 g of pure water was dropped and stirred at 60 ° C. for 3.5 hours. Resin particles A-3 having an average particle diameter of 370 nm were obtained. After sedimentation by centrifugation and removal of the supernatant, the operation of redispersing in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain 10 wt% resin particle dispersion B-3.

<Synthesis of resin-metal composite>
After adding 245 ml of pure water to the resin particle dispersion B-3 (90 ml), a 400 mM chloroplatinic acid aqueous solution (90 ml) was added, stirred at 30 ° C. for 3 hours, and allowed to stand at room temperature for 24 hours. Thereafter, the resin particles were precipitated by centrifugation, and the operation of removing the supernatant was repeated three times to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorption resin particle dispersion C-3.
Next, the C-3 (55 ml) was added to 3825 ml of pure water, and 132 mM dimethylamine borane aqueous solution (110 ml) was added dropwise with stirring at 3 ° C., followed by stirring at 3 ° C. for 1 hour. Thereafter, the mixture was stirred at 25 ° C. for 3 hours to obtain a resin-platinum composite D-3 having an average particle diameter of 382 nm. After D-3 was precipitated by centrifugation, the supernatant was removed, and the work of redispersing in pure water was repeated three times, and then purified by dialysis to remove impurities. Thereafter, the concentration was adjusted to obtain a 1 wt% resin-platinum composite dispersion E-3. The absorbance of the resin-platinum complex F-3 in E-3 was 0.70 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles of F-3 was 5 nm, and the supported amount of platinum was 38.5 wt%.

[Production Example 4]
<Synthesis of resin particles>
Aliquat 336 (manufactured by Aldrich) (1.50 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 49.50 g) and Divinylbenzene (DVB, 0.50 g) was added, and the mixture was stirred at 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 18.00 g of pure water was dropped and stirred at 60 ° C. for 3.5 hours. Resin particles A-4 having an average particle diameter of 430 nm were obtained. After sedimentation by centrifugation and removal of the supernatant, the operation of redispersing in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain a 10 wt% resin particle dispersion B-4.

<Synthesis of resin-metal composite>
After adding 245 ml of pure water to the resin particle dispersion B-4 (90 ml), a 400 mM chloroplatinic acid aqueous solution (90 ml) was added, stirred at 30 ° C. for 3 hours, and allowed to stand at room temperature for 24 hours. Thereafter, the resin particles were precipitated by centrifugation, and the operation of removing the supernatant was repeated three times to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to prepare 5 wt% platinum ion adsorbing resin particle dispersion C-4.
Next, the C-4 (55 ml) was added to 3825 ml of pure water, and 132 mM dimethylamine borane aqueous solution (110 ml) was added dropwise with stirring at 160 rpm and 3 ° C., followed by stirring at 3 ° C. for 1 hour. Thereafter, the mixture was stirred at 25 ° C. for 3 hours to obtain a resin-platinum composite D-4 having an average particle diameter of 454 nm. After D-4 was precipitated by centrifugation, the supernatant was removed, and the work of redispersing in pure water was repeated three times, and then purified by dialysis to remove impurities. Thereafter, the concentration was adjusted to obtain 1 wt% resin-platinum composite dispersion E-4. The absorbance of the resin-platinum complex F-4 in E-4 was 0.70 as a result of measurement according to the above method. Moreover, the average particle diameter of the platinum particles of F-4 was 5 nm, and the supported amount of platinum was 37.7 wt%.

[Production Example 5]
<Synthesis of gold colloid>
Add 500 ml of 1 mM chloroauric acid aqueous solution to a 500 ml three-necked round bottom flask and boil with vigorous stirring using a heating reflux apparatus. After boiling, add 25 ml of 38.8 mM sodium citrate aqueous solution. It was confirmed that the color changed to red. Heating was continued for 10 minutes while stirring, and then the mixture was allowed to cool at room temperature for about 30 minutes. The solution was filtered using a membrane filter having a pore size of 2 μm, transferred to an Erlenmeyer flask, and stored in a cool dark place. The average particle diameter of the produced particles was 12.3 nm. Moreover, the light absorbency was 1.32 as a result of measuring according to the said method.

[Reagents, etc.]
In Examples and Comparative Examples, the following reagents and the like were used.
Anti-influenza type A monoclonal antibody (7.15 mg / mL / PBS): manufactured by Adtec Corporation Anti-legionella polyclonal antibody Binding buffer a: 100 mM A boric acid solution was adjusted to pH≈3 with HCl.
Binding buffer b: A 100 mM boric acid solution was adjusted to pH≈8.5 with NaOH.
Blocking buffer a: 1% by weight bovine serum albumin solution was adjusted to pH≈5 with HCl.
Blocking buffer b: A 1% by weight bovine serum albumin solution was adjusted to pH≈8.5 with HCl.
Washing buffer: A 5 mM Tris solution was adjusted to pH≈8.5 with HCl.
Storage buffer: Sucrose was added to the wash buffer to a concentration of 10% by weight.
Influenza A positive control (APC): Influenza A virus inactivated antigen (manufactured by Adtech Co., Ltd.) was prepared by diluting 100 times with a sample treatment solution (manufactured by Adtech Co., Ltd.). The antigen concentration of APC corresponds to 5000 FFU / ml.
Negative control: Sample treatment solution (manufactured by Adtec Corporation)
AuNCP beads (1): resin-gold composite obtained in Production Example 1 AuNCP beads (2): resin-gold composite obtained in Production Example 2 PtNCP beads (1): resin-platinum composite obtained in Production Example 3 PtNCP beads (2): Resin-platinum composite obtained in Production Example 4

[Example 1]
(Joining process)
In a microtube [ibis (registered trademark; manufactured by ASONE) 2 mL], 0.1 mL of AuNCP beads (1) was added as a resin-metal complex, and binding buffer a0.9 mL was added. After thoroughly mixing by inversion mixing, an anti-influenza A monoclonal antibody is added, and the mixture is stirred for 3 hours at room temperature, and a labeled antibody-containing solution containing an anti-influenza A monoclonal antibody labeled with a resin-metal complex a-1 was obtained.

(Block process)
Next, the labeled antibody-containing solution a-1 is ice-cooled, centrifuged at 12000 rpm for 5 minutes, and the supernatant is removed. Then, the block buffer solution a1 mL is added to the solid residue, and it is applied for 10 to 20 seconds. Then, the mixture was subjected to ultrasonic dispersion treatment, and further stirred by inversion over 2 hours at room temperature to obtain a labeled antibody-containing liquid b-1.

(Cleaning process)
Next, after the labeled antibody-containing solution b-1 is ice-cooled, the mixture is centrifuged at 12000 rpm for 5 minutes, and the supernatant is removed. Then, 1 mL of a washing buffer is added to the solid residue, and the mixture is applied for 10 to 20 seconds. Then, ultrasonic dispersion treatment was performed. This operation was repeated three times to obtain a washing process.

(Save process)
Next, after cooling with ice, the mixture is centrifuged at 12000 rpm for 5 minutes, the supernatant is removed, 1 mL of a storage buffer is added to the solid residue, and ultrasonic dispersion treatment is performed for 10 to 20 seconds. As a result, a labeled antibody-containing solution c-1 was obtained.

<Evaluation method>
The evaluation was performed using a monochrome screen for influenza A evaluation (manufactured by Adtech), and the coloring levels after 5 minutes, 10 minutes and 15 minutes were compared. The color development level was determined using a color sample for gold colloid determination (manufactured by Adtech). In performance evaluation, the antigen used the 2-fold dilution series (1 times-1024 times) of influenza A positive control (APC).

<Evaluation procedure>
In each well of a 96-well plate, 3 μL of the labeled antibody-containing solution c-1 was added, and a 2-fold dilution series of APC (1-fold to 1024-fold, expressed as APC × 1 to APC × 1024, respectively) and a negative control, 100 μL was mixed. Next, 50 μL was added to the influenza A evaluation monochrome screen, and the color development level after 5 minutes, 10 minutes and 15 minutes was evaluated. The results are shown in Table 1. In addition, it means that a color development level is so high that a numerical value in Table 1 is large (color development is strong).

  From Table 1, it was confirmed that the labeled antibody-containing solution c-1 using AuNCP beads (1) showed good color development even with a 256-fold diluted antigen and had excellent labeling performance.

[Example 2]
A labeled antibody-containing solution c-2 was prepared in the same manner as in Example 1 except that the AuNCP beads (2) were used instead of the AuNCP beads (1), and the color development level was evaluated. The results are shown in Table 2.

  From Table 2, it was confirmed that the labeled antibody-containing solution c-2 using AuNCP beads (2) showed good color development even with a 256-fold diluted antigen and had excellent labeling performance.

[Example 3]
A labeled antibody-containing solution c-3 was prepared in the same manner as in Example 1 except that PtNCP beads (1) were used instead of AuNCP beads (1), and the color development level was evaluated. The results are shown in Table 3.

  From Table 3, it was confirmed that the labeled antibody-containing solution c-3 using the PtNCP beads (1) showed good color development even with 512-fold diluted antigen and had excellent labeling performance.

[Example 4]
A labeled antibody-containing solution c-4 was prepared in the same manner as in Example 1 except that the PtNCP beads (2) were used in place of the AuNCP beads (1), and the color development level was evaluated. The results are shown in Table 4.

  From Table 4, it was confirmed that the labeled antibody-containing solution c-4 using the PtNCP beads (2) showed good color development even with respect to the antigen diluted 1024 times and had excellent labeling performance.

[Example 5]
(Joining process)
To a microtube [ibis (registered trademark; manufactured by ASONE) 2 mL], 0.1 mL of PtNCP beads (1) was added as a resin-metal complex, and binding buffer a0.9 mL was added. After thoroughly mixing by inversion mixing, an anti-legionella polyclonal antibody is added, and the mixture is agitated by inversion over 3 hours at room temperature, and a labeled antibody-containing solution a-5 containing an anti-legionella polyclonal antibody labeled with a resin-metal complex is obtained. Obtained.

(Block process)
Next, after the labeled antibody-containing solution a-5 is ice-cooled, the mixture is centrifuged at 12000 rpm for 5 minutes, and the supernatant is removed. Then, the block buffer solution a1 mL is added to the solid residue, and it is applied for 10 to 20 seconds. Then, the mixture was subjected to ultrasonic dispersion treatment, and further stirred by inverting at room temperature for 2 hours to obtain a labeled antibody-containing liquid b-5.

(Cleaning process)
Next, after cooling the labeled antibody-containing solution b-5 with ice and centrifuging at 12000 rpm for 5 minutes and removing the supernatant, 1 mL of a washing buffer solution is added to the solid residue, and the solution is applied for 10 to 20 seconds. Then, ultrasonic dispersion treatment was performed. This operation was repeated three times to obtain a washing process.

(Save process)
Next, after cooling with ice, the mixture is centrifuged at 12000 rpm for 5 minutes, the supernatant is removed, 1 mL of a storage buffer is added to the solid residue, and ultrasonic dispersion treatment is performed for 10 to 20 seconds. To obtain a labeled antibody-containing solution c-5.

(Padded)
The labeled antibody-containing solution C-5 was diluted 4-fold, impregnated in a 30 cm × 1 cm glass fiber pad (manufactured by Merck Millipore), and dried at 20 ° C. for 60 minutes to obtain a labeled antibody-containing pad.

<Evaluation method>
The evaluation was performed using a reaction strip in which an anti-Legionella polyclonal antibody was immobilized on a nitrocellulose membrane (manufactured by Merck Millipore), and the color development levels after 5 minutes, 10 minutes, and 15 minutes were compared. The color development level was determined using a color sample for gold colloid determination (manufactured by Adtech). In the performance evaluation, a two-fold dilution series of Legionella antigen (concentration: 2.0 × 10 ⁵ cfu / mL) was used as the antigen.

<Evaluation procedure>
In each well of a 96-well plate, 100 μL each of a 2-fold dilution series of APC (1-fold to 32-fold, each represented as PC × 1 to PC × 32) and a negative control was mixed. Next, 100 μL of a buffer solution (100 mM Tris-HCl, 1% Triton X-100) was mixed, 100 μL of the mixed solution was added to the reaction strip, and the color development level after 15 minutes was evaluated. As a comparative example, a labeled antibody-containing pad labeled with the gold colloid prepared in Preparation Example 5 was used instead of the PtNCP beads (1). The results are shown in Table 5.

A labeled antibody-containing solution containing an anti-Legionella polyclonal antibody labeled with gold colloid was prepared by the following procedure.
100 μg of anti-Legionella polyclonal antibody was mixed with 1 ml of gold colloid (OD = 10) and stirred at room temperature for about 3 hours to bind the antibody to the gold colloid. The bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the colloidal gold surface. The gold colloid-labeled antibody was prepared by centrifuging at 12000 rpm and 4 ° C. for 5 minutes and then suspending in a buffer containing 0.2% bovine serum albumin.

  From Table 5, the labeled antibody-containing pad using the PtNCP beads (1) shows good color development even with a 32-fold diluted antigen, which is superior to the labeled antibody-containing pad using colloidal gold. It was confirmed to have performance.

  As mentioned above, although embodiment of this invention was described in detail for the purpose of illustration, this invention is not restrict | limited to the said embodiment.

DESCRIPTION OF SYMBOLS 10 ... Resin particle, 20 ... Metal particle, 30 ... Encapsulated metal particle, 40 ... Partially exposed metal particle, 50 ... Surface adsorbed metal particle, 60 ... Surface layer part, 100 ... Resin-metal composite, 110 ... Membrane, 120 ... Sample addition part, 130 ... determination part, 131 ... capture ligand, 140 ... liquid absorption part, 150 ... labeled antibody, 160 ... influenza virus, 170 ... complex, 200 ... test strip

Claims (9)

An immunoassay method for detecting or quantifying viruses or bacteria contained in a sample,
Using a lateral flow type chromatographic test strip comprising a membrane and a determination part having a capture ligand that specifically binds to the virus or bacteria immobilized on the membrane, the following steps (I) to (III);
Step (I): The virus or bacteria contained in the sample and an antibody that specifically binds to the virus or bacteria are labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles. A step of contacting the labeled antibody,
Step (II): A step of bringing the complex containing a virus or bacteria and a labeled antibody formed in Step (I) into contact with a capture ligand in the determination unit,
Step (III): a step of measuring a color intensity derived from light energy absorption by localized surface plasmon resonance and electronic transition of the resin-metal complex,
An immunoassay method comprising the steps of:   The immunoassay method according to claim 1, wherein the metal particles are gold particles or platinum particles. The metal particles in the resin-metal composite are:
First particles having a portion exposed outside the resin particles;
Second particles entirely encapsulated in the resin particles;
Contains
The immunoassay according to claim 1 or 2, wherein at least some of the first particles and the second particles are three-dimensionally distributed in a surface layer portion of the resin particles. Method.   The immunoassay method according to any one of claims 1 to 3, wherein the resin particle is a polymer particle having a substituent capable of adsorbing metal ions in its structure.   The immunoassay method according to any one of claims 1 to 4, wherein an average particle diameter of the metal particles is in a range of 1 to 80 nm.   The immunoassay method according to any one of claims 1 to 5, wherein an average particle diameter of the resin-metal complex is in a range of 50 to 1000 nm.   The immunoassay method according to any one of claims 1 to 6, wherein the antibody is an anti-influenza A monoclonal antibody. An immunoassay kit for detecting or quantifying viruses or bacteria contained in a sample using a lateral flow type chromatographic test strip,
A lateral flow type chromatographic test strip including a membrane, and a determination unit in which a capture ligand that specifically binds to the virus or bacteria is immobilized on the membrane;
A detection reagent comprising a labeled antibody in which an antibody that specifically binds to the virus or bacteria is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles;
A kit for immunoassay for detecting or quantifying viruses or bacteria containing. A lateral flow type chromatographic test strip for detecting or quantifying viruses or bacteria contained in a sample,
A membrane,
A determination unit in which a capture ligand that specifically binds to the virus or bacteria is immobilized on the membrane in the direction in which the sample develops,
A labeled antibody obtained by labeling an antibody that specifically binds to the virus or bacterium with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle is included upstream of the determination unit. A reaction part;
Lateral flow type chromatographic test strip.